Influence of mismatched and bulged nucleotides on SNP-preferential RNase H cleavage of RNA-antisense gapmer heteroduplexes

Abstract

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.

Figure 1

A map of RNA/gapmer heteroduplex. (A) General SNP positioning in the context of a gapmer structure. (B) Different arrangements of mismatches applied in the study.

Figure 2

The influence of C-C mismatch position on yield of RNase H cleavage of RNA 692. T1 – RNase T1 ladder, C- control RNA (without ASO), 15–20 – gapmers b15-b20, M- gapmer bKM complementary to mutant RNA 692, order of the gapmers on gel corresponds with changing C-dC mismatch position along the gap. b15 – C-dC at position 3/13 from 5′ end of the gapmer, b20 – C-dC at position 10/13 from 5′ end of the gapmer. This gel image was cropped and rearranged to present data of interest. Original gel image is included in Supplementary Materials as Fig. S14.

Figure 3

Comprehensive results of in vitro activity of gapmers designed to APP RNA 692 and RNA 693 targets. (A) Target APP mRNA sequence containing sites of C-to-G (RNA692) and A-to-G (RNA693) SNPs and designed to them antisense gapmers. SNPs in RNA strand are bolded and underlined, codons in which they occur are marked by red frame. Modified nucleotides within gapmers are marked by colours: blue-LNA, green-2′-O-MeRNA, bolded black- DNA, red-mismatched nucleotides, underlined-SNP site. (B) RNase H in vitro assay results for APP RNA692 target, showing the influence of arrangements of mismatched nucleotides within 13, 15 and 17 nt-long RNA/ASO duplexes on RNase H cleavage. Blue frames mark gapmers which cause selective degradation of Mut RNA. (C) Thermodynamic parameters of wild type and mutant RNA/gapmer duplexes containing mismatches. Oligonucleotides, which differentiated two alleles cleavage yields in RNase H assay were only measured. Parameters for more stable duplex of the two (wild type/ASO or mutant/ASO) are bolded. (D) RNase H in vitro assay results for APP RNA693 target, showing the influence of arrangements of mismatched nucleotides within 13 nt-long RNA/ASO duplexes on RNase H cleavage. Blue frames mark gapmers which cause selective degradation of Mut RNA.

Figure 4

Activity of selected gapmers, designed to APP RNA 692 and RNA 693 targets, in HeLa cells. (A) Results of ASOs transfection to HeLa cells. The EC₅₀ values for gapmers tested in presence of WT and mutant alleles were estimated based on dose-response curves fitted to experimental data in Origin Lab 8.0 software (B). The data were gathered from at least three separate experiments involving five concentrations in the range 10–100 nM. Comparison of EC₅₀ values for gapmers between WT and Mutant target RNA alleles allowed to evaluate allele-preference for RNA cleavage of tested gapmers. Two last columns of the table present WT/Mut ratio of gapmer selectivity and the selectivity of mismatched gapmers relative to complementary gapmer. (B) Dose-response curves for selected antisense gapmers. Upper charts – ASO targeting APP Flemish variant (RNA692): bKM - referenced ASO gapmer (mutant complementary to RNA692 target); b8, b8-a – gapmers presenting selectivity to Mut RNA692 in HeLa cells; Lower charts – ASO targeting APP Arctic variant (RNA693) eKM - referenced ASO gapmer (mutant complementary to RNA693 target); e3 – gapmer selective to Mut RNA693 target in HeLa cells; for statistics see Supplementary data.

Figure 5

Comprehensive results of in vitro activity of gapmers designed to APP RNA 717 target (APP London variant). (A) Target APP mRNA sequence containing site of G-to-A (RNA717) SNP and designed to it antisense gapmers. SNP in RNA strand is bolded and underlined, codon in which it occurs is marked by red frame. Modified nucleotides within gapmers are marked by colours: blue-LNA, green-2′-O-MeRNA, bolded black- DNA, red-mismatched nucleotides, underlined-SNP site. (B) RNase H in vitro assay results for APP RNA717 target, showing the influence of arrangements of mismatched nucleotides in 13 nt-long RNA/ASO duplexes on RNase H cleavage. Blue frames mark gapmers which cause preferential cleavage of Mut RNA. Red frames mark gapmer which cause preferential cleavage of WT RNA. (C) Thermodynamic parameters of APP 717 wild type and mutant RNA/gapmer duplexes containing mismatches. Oligonucleotides, which differentiated two alleles cleavage yields in RNase H assay were only measured. Parameters for more stable duplex of the two (wild type/ASO or mutant/ASO) are bolded.

Figure 6

Activity of selected gapmers, designed to APP 717 target, in HeLa cells. (A) Results of ASOs transfection to HeLa cells. The EC₅₀ values for gapmers tested in presence of WT and mutant alleles were estimated based on dose-response curves fitted to experimental data in Origin Lab 8.0 software (for statistics see Supplementary data). The data were gathered from at least three separate experiments involving five concentrations in the range 10–100nM. Comparison of EC₅₀ values for gapmers between WT and Mutant target RNA alleles allowed to evaluate allele-preference for RNA cleavage of tested gapmers. Two last columns of the table present WT/Mut ratio of gapmer selectivity and the selectivity of mismatched gapmers relative to complementary gapmer dKM. Plots present dose-response curves for selected antisense gapmers. (B) Dose-response curves for selected antisense gapmers. dKM - referenced ASO gapmer (mutant complementary to RNA717 target); d14 – ASO gapmer presenting SNP preference in HeLa cells, however not in the RNase H assay in vitro. Although the both models for d14 gapmer were statistically insignificant (P > 0.05), validation experiment confirmed SNP-preferential cleavage in concentrations determined from the curves (Fig. S13).

Figure 7

Comprehensive results of in vitro activity of gapmers designed to SNCA RNA 46 and RNA 53 targets. (A) Target SNCA mRNA sequence containing sites of two G-to-A SNPs (RNA46 and RNA53) and designed to them antisense gapmers. SNPs in RNA strand are bolded and underlined, codons in which they occur are marked by red frame. Modified nucleotides within gapmers are marked by colours: blue-LNA, green-2′-O-MeRNA, bolded black- DNA, red-mismatched nucleotides, underlined-SNP site. (B) RNase H in vitro assay results for SNCA RNA46 target, showing the influence of arrangements of mismatched nucleotides in RNA/ASO duplexes on RNase H cleavage. Blue frames mark gapmers which cause selective cleavage of Mut RNA, red frames mark gapmers which preferentially cause wild type RNA cleavage. (C) RNase H in vitro assay results for SNCA RNA53 target, showing the influence of arrangements of mismatched nucleotides in RNA/ASO duplexes on RNase H cleavage. Blue frames mark gapmers which cause selective cleavage of Mut RNA, red frames mark gapmers which preferentially cause wild type RNA cleavage. (D) Thermodynamic parameters of SNCA wild type and mutants RNA/gapmer duplexes containing mismatches. Oligonucleotides, which differentiated two alleles cleavage yields in RNase H assay were only measured. Parameters for more stable duplex of the two (wild type/ASO or mutant/ASO) are bolded.

Figure 8

Activity of selected gapmers, designed to SNCA RNA 46 and RNA 53 targets, in HeLa cells. (A) Results of ASOs transfection to HeLa cells. The EC₅₀ values for gapmers tested in presence of WT and mutant alleles were estimated based on dose-response curves fitted to experimental data in Origin Lab 8.0 software (for statistics see Supplementary data). The data were gathered from at least three separate experiments involving five concentrations in the range 10–100nM. Comparison of EC₅₀ values for gapmers between WT and Mutant target RNA alleles allowed to evaluate allele-preference for cleavage of tested gapmers. Two last columns of the table present WT/Mut ratio of gapmer selectivity and the selectivity of mismatched gapmers relative to complementary gapmers (kKM for RNA 46 target and hKM for RNA 53 target). (B) Dose-response curves for antisense gapmers causing preferential cleavage of Mut 46 RNA (kKM) and Mut 53 RNA (h2).

Figure 9

Activity of gapmers designed to SOD1 RNA 4 target in vitro and in HeLa cells. (A) Target SOD1 mRNA sequence containing site of C-to-U SNP (RNA4) and designed to it antisense gapmers. SNP in RNA strand is bolded and underlined, codon in which it occur is marked by red frame. Modified nucleotides within gapmers are marked by colours: blue-LNA, green-2′-O-MeRNA, bolded black- DNA, red-mismatched nucleotides, underlined-SNP site. (B) RNase H in vitro assay results for SOD1 RNA4 target, showing the influence of arrangements of mismatched nucleotides in RNA/ASO duplexes on RNase H cleavage. Red frames mark gapmers which cause undesirable effect of preferential wild type RNA cleavage. Blue frames mark gapmers which cause selective degradation of Mut RNA. (C) Results of ASOs transfection to HeLa cells. The EC₅₀ values for gapmers tested in presence of WT and mutant alleles were estimated based on dose-response curves fitted to experimental data in Origin Lab 8.0 software (for statistics see Supplementary data). The data were gathered from at least three separate experiments involving five concentrations in the range 10–100nM. Comparison of EC₅₀ values for gapmers between WT and Mutant target RNA alleles allowed to evaluate allele-preference for cleavage of tested gapmers. Two last columns of the table present WT/Mut ratio of gapmer selectivity and the selectivity of mismatched gapmers relative to complementary gapmer aKM.

Figure 10

Activity of gapmers designed to SCA3 RNA target in vitro and in HeLa cells. (A) Target SCA3 mRNA sequence containing site of G-to-C SNP and designed to it antisense gapmers. SNP in RNA strand is bolded and underlined, codon in which it occur is marked by red frame. Modified nucleotides within gapmers are marked by colours: blue-LNA, green-2′-O-MeRNA, bolded black- DNA, red-mismatched nucleotides, underlined-SNP site. (B) RNase H in vitro assay results for SCA3 RNA target, showing the influence of arrangements of mismatched nucleotides in 13 nt-long RNA/ASO duplexes on RNase H cleavage. Blue frames mark gapmers which cause preferential cleavage of Mut RNA. (C) Results of ASOs transfection to HeLa cells. The EC₅₀ values for gapmers tested in presence of WT and mutant alleles were estimated based on dose-response curves fitted to experimental data in Origin Lab 8.0 software (for statistics see Supplementary data). The data were gathered from at least three separate experiments involving up to five concentrations in the range 10–100 nM. Comparison of EC₅₀ values for gapmers between WT and Mutant target RNA alleles allowed to evaluate allele-preference for cleavage of tested gapmers. Two last columns of the table present WT/Mut ratio of gapmer selectivity and the selectivity of mismatched gapmers relative to complementary gapmer fKM.